Inhibition of nucleic acid polymerases by endonuclease V-cleavable circular oligonucleotide ligands

ABSTRACT

Provided are methods and compositions for activating oligonucleotide aptamer-deactivated DNA polymerases, comprising cleaving the aptamer by endonuclease V enzymatic activity to reduce or eliminate binding of the oligonucleotide aptamer to the DNA polymerase, thereby activating DNA synthesis activity of the DNA polymerase in a reaction mixture. Mixtures for use in methods of the invention are also provided. The oligonucleotide aptamers of the present invention are circular and comprise one or more deoxyinosine nucleotides providing for aptamer-specific recognition and cleavage of the circular aptamer by the endonuclease V enzymatic activity. Exemplary oligonucleotide aptamers, mixtures and methods employing endonuclease V enzymatic activity are provided. The methods can be practiced using kits comprising a DNA polymerase-binding oligonucleotide aptamer and at least one endonuclease V enzymatic activity having oligonucleotide aptamer-specific recognition to provide for specific cleavage of the aptamer by the endonuclease V enzymatic activity.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No. 16/210,608, filed Dec. 5, 2018, which claims the benefit of U.S. Provisional Patent Application No. 62/595,453, filed Dec. 6, 2017, the disclosures of which are herein incorporated by reference in their entirety.

INCORPORATION OF SEQUENCE LISTING

The contents of the text file named “0102384-002US1 Sequence Listing.txt,” which was created on Jun. 12, 2020, and is 8 KB in size, are hereby incorporated by reference in their entirety.

BACKGROUND

Aspects of the present invention relate generally to improved methods of blocking DNA polymerase activity with oligonucleotide aptamers at low reaction temperatures, and restoring the enzyme activity upon raising the reaction temperature (e.g., hot-start methods).

DNA polymerases are enzymes used for synthesis of DNA strands by primer extension, wherein the polymerase-catalyzed DNA synthesis may be initiated by oligonucleotide primers hybridized to a complementary template DNA. Initiating DNA synthesis from this template-hybridized primer, DNA polymerases create complementary DNA strands in the presence of corresponding nucleotide 5′-triphosphates. Sequence specificity of nucleotide polymerization, when the oligonucleotide primers bind exclusively to the desired sites and nowhere else, is an important requirement in many applications wherein DNA synthesis is used. However, the efficiency and fidelity of DNA synthesis can be reduced when primers hybridize to non-complementary DNAs, leading to synthesis of incorrect DNA sequences.

Many so-called ‘Hot Start’ methods have been developed to avoid incorrect primer extension products (e.g., see Paul N., et al. (2010), for review). One of the most common techniques is based on use of oligonucleotide aptamers (Jayasena, S. D., 1999). Aptamers offer a number of advantages over other reported methods. Using a method of molecular evolution (SELEX), they can be quickly engineered in a test tube and then readily and inexpensively manufactured by chemical synthesis. Ideally, an aptamer should: (i) completely block DNA polymerase at low temperatures, and (ii) provide no blockage effect at the desired elevated reaction temperature. Unfortunately, this is very difficult, if not impossible to achieve, and the aptamer structure usually represents a compromise between these two key requirements. New methods, therefore, are needed to improve control of aptamer activity in reaction mixtures containing DNA polymerases.

Particular aspects provide methods of activating an aptamer-inactivated DNA polymerase, comprising: providing a reaction mixture suitable for DNA synthesis, the reaction mixture comprising (i) a DNA polymerase, (ii) an endonuclease V-cleavable circular oligonucleotide aptamer that binds to the DNA polymerase, wherein the oligonucleotide aptamer is present in an amount effective to inhibit DNA synthesis activity of the DNA polymerase in the reaction mixture, and (iii) an endonuclease V enzymatic activity; and cleaving the circular aptamer by the endonuclease V enzymatic activity to reduce or eliminate binding of the circular oligonucleotide aptamer to the DNA polymerase, thereby activating the DNA synthesis activity of the DNA polymerase, to increase DNA synthesis in the reaction mixture. In the methods, cleaving may be facilitated using a reaction temperature that facilitates both DNA polymerase activity and the endonuclease V enzymatic activity. In the methods, cleaving may be facilitated by increasing the temperature of the reaction mixture from a first temperature to a second temperature that more strongly facilitates the endonuclease V enzymatic activity. In the methods, providing a reaction mixture suitable for DNA synthesis may comprise dissolving a dried form of at least one of said (i) DNA polymerase, (ii) endonuclease V-cleavable circular oligonucleotide aptamer, and (iii) endonuclease V enzymatic activity into an aqueous solution. DNA synthesis in the methods may result in DNA amplification in the reaction mixture (e.g., wherein the DNA amplification comprises PCR) DNA synthesis in the methods may comprise an isothermal amplification reaction. The methods may comprise detecting the presence of a target DNA and/or measuring an amount of a target DNA in the reaction mixture. In the methods, the circular oligonucleotide aptamer may comprise one or more deoxyinosine nucleotides. In the methods, the circular oligonucleotide aptamer may have a stem-loop structure, wherein one or more deoxyinosine nucleotides may be incorporated into the stem segment of the stem-loop structure, and/or wherein one or more deoxyinosine nucleotides may be incorporated into the loop segment of the stem-loop structure. In the methods, the loop of the stem-loop structure, may, for example, comprise a nucleotide sequence selected from the group consisting of 5′-TTCITAGCGTTT-3′ (SEQ ID NO:19), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20), 5′-TTCTTAICGTTT-3′ (SEQ ID NO:21), 5′-TTCIIAGCGTTT-3′ (SEQ ID NO:22), 5′-TTCITAICGTTT-3′ (SEQ ID NO:23), 5′-TTCTIAICGTTT-3′ (SEQ ID NO:24), and 5′-TTCIITAICGTTT-3′ (SEQ ID NO:25). In the methods, the loop of the stem-loop structure may, for example, comprise one of the nucleotide sequences 5′-TTCITAGCGTTT-3′ (SEQ ID NO:19), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20) or 5′-TTCTTAICGTTT-3′ (SEQ ID NO:21). In the methods, the endonuclease V enzymatic activity may comprise, for example, Thermotoga maritima endonuclease V enzymatic activity.

Additional aspects provide kits for activating an aptamer-inactivated DNA polymerase, comprising: an endonuclease V enzymatic activity; and a DNA polymerase-binding circular oligonucleotide aptamer cleavable by an endonuclease V enzymatic activity. In the kits, the circular oligonucleotide aptamer may comprise one or more deoxyinosine nucleotides. In the kits, the circular oligonucleotide aptamer may comprise a stem-loop structure. In the kits, one or more deoxyinosine nucleotides may be located in the stem segment of the stem-loop structure, and/or one or more deoxyinosine nucleotides may be located in the loop segment of the stem-loop structure. In the kits, the loop of the stem-loop structure, may, for example, comprise a nucleotide sequence selected from the group consisting of 5′-TTCITAGCGTTT-3′ (SEQ ID NO:19), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20), 5′-TTCTTAICGTTT-3′ (SEQ ID NO:21), 5′-TTCIIAGCGTTT-3′ (SEQ ID NO:22), 5′-TTCITAICGTTT-3′ (SEQ ID NO:23), 5′-TTCTIAICGTTT-3′ (SEQ ID NO:24), and 5′-TTCIITAICGTTT-3′ (SEQ ID NO:25). In the kits, the loop of the stem-loop structure may, for example, comprise one of the nucleotide sequences 5′-TTCITAGCGTTT-3′ (SEQ ID NO:19), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20) or 5′-TTCTTAICGTTT-3′ (SEQ ID NO:21). In the kits, the endonuclease V enzymatic activity may comprise, for example, Thermotoga maritima endonuclease V enzymatic activity.

Further aspects provide reaction mixtures for use in a method of DNA synthesis, which reaction mixture comprises: a DNA polymerase; an endonuclease V-cleavable circular oligonucleotide aptamer that binds reversibly to the DNA polymerase, wherein the circular oligonucleotide aptamer is present in an amount effective to inhibit DNA synthesis activity of the DNA polymerase in the reaction mixture; and an endonuclease V enzymatic activity capable of cleaving (or effective to cleave) the circular oligonucleotide aptamer to reduce or eliminate binding of the circular oligonucleotide aptamer to the DNA polymerase, thereby activating the DNA synthesis activity of the DNA polymerase. In the reaction mixtures, the DNA polymerase activity and/or the endonuclease V enzymatic activity may be temperature-dependent. In the reaction mixtures, the endonuclease V enzymatic activity may increase when the reaction mixture is heated from a first temperature value to a second temperature value that promotes the endonuclease V enzymatic activity. In the reaction mixtures, the DNA polymerase, oligonucleotide aptamer, and endonuclease V enzymatic activity may be in a dried state. In the reaction mixtures, the circular oligonucleotide aptamer may comprise one or more deoxyinosine nucleotides. In the reaction mixtures, the circular oligonucleotide aptamer may comprise a stem-loop structure. In the reaction mixtures, one or more deoxyinosine nucleotides may be located in the stem segment of the stem-loop structure, and/or one or more deoxyinosine nucleotides may be located in the loop segment of the stem-loop structure. In the reaction mixtures, the loop of the stem-loop structure, may, for example, comprise a nucleotide sequence selected from the group consisting of 5′-TTCITAGCGTTT-3′ (SEQ ID NO:19), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20), 5′-TTCTTAICGTTT-3′ (SEQ ID NO:21), 5′-TTCIIAGCGTTT-3′ (SEQ ID NO:22), 5′-TTCITAICGTTT-3′ (SEQ ID NO:23), 5′-TTCTIAICGTTT-3′ (SEQ ID NO:24), and 5′-TTCITAICGTTT-3′ (SEQ ID NO:25). In the reaction mixtures, the loop of the stem-loop structure may, for example, comprise one of the nucleotide sequences 5′-TTCITAGCGTTT-3′ (SEQ ID NO:19), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20) or 5′-TTCTTAICGTTT-3′ (SEQ ID NO:21). In the reaction mixtures, the endonuclease V enzymatic activity may comprise, for example, Thermotoga maritima endonuclease V enzymatic activity. The reaction mixtures may further comprise one or more of dATP, dCTP, dGTP, and/or dTTP, and/or Mg²⁺ ion.

Yet further aspects provide circular oligonucleotide aptamers, comprising a nucleic acid sequence that forms a hairpin structure, having a stem and a loop portion, that binds to a DNA polymerase, wherein the stem and/or the loop portion comprises one or more deoxyinosine nucleotides, and wherein the loop portion comprises a nucleotide sequence 5′-TTCTTAGCGTTT-3′ (SEQ ID NO:18) that may be substituted with deoxyinosine at one or more of positions 4, 5, and 7. In the oligonucleotide aptamers, the loop portion may comprise the nucleotide sequence 5′-TTCTTAGCGTTT-3′ (SEQ ID NO:18). In the oligonucleotide aptamers, one or more deoxyinosine nucleotides may be located in the stem segment of the stem-loop structure, and/or one or more deoxyinosine nucleotides may be located in the loop segment of the stem-loop structure. In the oligonucleotide aptamers, the loop of the stem-loop structure, may, for example, comprise a nucleotide sequence selected from the group consisting of 5′-TTCITAGCGTTT-3′ (SEQ ID NO:19), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20), 5′-TTCTTAICGTTT-3′ (SEQ ID NO:21), 5′-TTCIIAGCGTTT-3′ (SEQ ID NO:22), 5′-TTCITAICGTTT-3′ (SEQ ID NO:23), 5′-TTCTIAICGTTT-3′ (SEQ ID NO:24), and 5′-TTCIITAICGTTT-3′ (SEQ ID NO:25). In the circular oligonucleotide aptamers, the loop of the stem-loop structure may, for example, comprise one of the nucleotide sequences 5′-TTCITAGCGTTT-3′ (SEQ ID NO:19), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20) or 5′-TTCTTAICGTTT-3′ (SEQ ID NO:21). In the oligonucleotide aptamers, the one or more deoxyinosine nucleotides may render the circular oligonucleotide aptamer cleavable by an endonuclease V enzymatic activity (e.g., wherein the endonuclease V enzymatic activity may comprise Thermotoga maritima endonuclease V enzymatic activity).

Still further aspects provide compositions comprising a DNA polymerase complexed with a circular oligonucleotide aptamer as disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows, according to particular exemplary aspects, a portion of a human β2-microglobulin gene sequence (SEQ ID NO:4), forward and reverse primers (SEQ ID NOS:1-2, respectively) and a 22-mer fluorescent probe (SEQ ID NO:3), which were used in exemplary 5′-nuclease PCR assays of the present invention from which exemplary results are shown in FIGS. 3 and 5. The primers and probe are shown aligned with an amplified β2-microglobulin fragment sequence in 5′→3′ orientation as indicated.

FIG. 2 shows, according to particular exemplary aspects, structures of circular stem-loop deoxyribonucleotide aptamers (shown at the right column of FIG. 2 under “Aptamer in circular form”) used in exemplary 5′-nuclease PCR assays of the present invention from which exemplary results are shown in FIGS. 3A and 3B. The central column of FIG. 2 shows linear oligonucleotide sequences used to prepare the corresponding circular aptamers SEQ ID NOS:6-11 as described herein in Example 2. The aptamer SEQ ID NO:5 (shown at the top right of FIG. 2) is a noncircular analog of aptamer SEQ ID NO:6 used in experiments to obtain the results shown in FIGS. 3A and 3B illustrating the effect of circularization on the aptamer activity of deactivating DNA polymerase. The symbol “p” indicates 5′-phosphate moiety. The symbol “I” indicates the presence of deoxyriboinosine nucleotides in aptamers SEQ ID NOS:7-11 that were used in the experiments with endonuclease V. Aptamers SEQ ID NOS:7 and 8 incorporate deoxyinosine within the 5′-3′ duplex strand whereas aptamers SEQ ID NOS:9-11 incorporate deoxyinosine within the 3′-5′ duplex strand. Arrows point to the expected endonuclease V cleavage positions.

FIGS. 3A and 3B show, according to particular exemplary aspects, the results of fluorescence monitoring of reaction mixtures during PCR (real-time curves) in the presence of the individual aptamers listed in FIG. 2. Sequences of the amplified β2-microglobulin template, primers and 22-mer FRET probe used in these PCR assays are as shown in FIG. 1. Dashed lines are real-time curves obtained in the absence of any aptamer. Experiments were conducted in the absence (FIG. 3A) or presence (FIG. 3B) of T. maritima endonuclease V. Experimental details are provided below in “Example 3.”

FIG. 4 shows, according to particular exemplary aspects, structures of four additional circular stem-loop deoxyribonucleotide aptamers SEQ ID NOS:12-15 (right column of FIG. 4), which are derivatives of the circular aptamer SEQ ID NO:6 of FIG. 2 and incorporate deoxyinosine nucleotide (“I”) in the loop portion at various indicated positions. These aptamers were used in the 5′-nuclease PCR assays from which exemplary results are shown in FIGS. 5A and 5B. The central column of FIG. 4 shows linear oligonucleotide sequences used to prepare the corresponding circular aptamers SEQ ID NOS:12-15 (shown at the right column of FIG. 4 under “Aptamer in circular form”), as described herein in Example 2. The symbol “p” indicates a 5′-phosphate moiety. Arrows point to the expected endonuclease V cleavage positions.

FIGS. 5A and 5B show, according to particular exemplary aspects, the results of fluorescence monitoring of reaction mixtures during PCR (real-time curves) in the presence of the individual circular aptamers listed in FIG. 4. Sequences of the amplified β2-microglobulin template, primers and 22-mer FRET probe used in the assays are as shown in FIG. 1. Dashed lines are real-time curves obtained in the absence of any aptamer. Experiments were conducted in the absence (FIG. 5A) or presence (FIG. 5B) of T. maritima endonuclease V. Experimental details are provided herein below under “Example 3.”

FIG. 6 shows, according to particular exemplary aspects, a reaction scheme used in the assays of FIGS. 7A through 7F to detect and measure DNA polymerase activity. The depicted hairpin-like fluorescent probe SEQ ID NO:16 was designed to have a G/C-rich stem in the duplex segment and a 5′- . . . GAA . . . hairpin-stabilizing loop to provide for use at elevated temperatures (e.g., up to 60-65° C.) (e.g., see Yoshizawa S., et al, 1994). Extension of this hairpin-like probe in a reaction buffer in the presence of deoxyribonucleoside 5′-triphosphates (dNTPs) and a DNA polymerase results in a fluorescent signal that directly correlates with the polymerase activity in the reaction.

FIGS. 7A through 7F show, according to particular exemplary aspects, endonuclease V-induced activation of Taq (FIG. 7D), Phusion® (FIG. 7B), Q5® (FIG. 7C), Vent® (FIG. 7A), Deep Vent® (FIG. 7E) and Bst large fragment (FIG. 7F) DNA polymerases that were initially deactivated (i.e., “inhibited” or “blocked”) by the presence of aptamer SEQ ID NO:12 (♦ curves). FIGS. 7A through 7F also show the change of fluorescence with time in the absence of endonuclease V for the aptamer-blocked (∘ curves) and unblocked (□ curves) DNA polymerase. The DNA polymerase activity was monitored by extension of the self-priming hairpin-like fluorescent probe SEQ ID NO:16 (see FIG. 6), which was present in the reaction mixture with all four dNTPs in a magnesium-containing buffer. In all cases experiments were performed at 60 or 65° C., as indicated in each Figure. The structure of aptamer SEQ ID NO:12 is shown in FIG. 4, and details of the experimental setup, results analysis and conclusions are provided below in “Example 4.”

DETAILED DESCRIPTION Definitions

Terms and symbols of biochemistry, nucleic acid chemistry, molecular biology and molecular genetics used herein follow those of standard treatises and texts in the field (e.g., Sambrook, J., et al, 1989; Kornberg, A., and Baker, T., 1992; Gait, M. J., ed., 1984; Lehninger, A. L., 1975; Eckstein F., ed., 1991, and the like). To facilitate understanding of particular exemplary aspects of the invention, a number of terms are discussed below.

In particular aspects, “aptamer” or “oligonucleotide aptamer” refers herein to an oligonucleotide that is capable of binding to a DNA polymerase and blocking its DNA synthesis enzymatic activity. The aptamers can be linear circular molecules and/or capable to form secondary structures such as hairpin or stem-loop structures, etc. Examples of aptamers and methods of selection (design) can be found, for instance, in Yakimovich, O. Yu., et al. (2003); Jayasena, S. D. (1999); U.S. Pat. No. 5,693,502, issued to Gold, L. and Jayasena, S. D., which are incorporated here by reference. As used herein, the term “circular” refers to oligonucleotide that forms a closed loop and has no ends. The phrase “aptamer, that binds to the DNA polymerase, in an amount sufficient to inhibit DNA synthesis activity of the DNA polymerase,” as used herein, means that the DNA synthesis activity of the DNA polymerase is at least partially inhibited (e.g., inhibited to a level in the range of from about 1% to about 99.99%). Any level of aptamer inhibition of the DNA synthesis activity of the DNA polymerase can provide an advantage for DNA synthesis, and thus according to particular preferred hot start aspects of the present invention, the DNA synthesis activity of the DNA polymerase is substantially inhibited (e.g., inhibited to a level in the range of about 80% to 99.99%, or to any subrange or level therein), or completely (100%) inhibited, providing an advantage over other ‘hot start’ technologies (e.g., Paul N., et al., 2010). Likewise, in the disclosed methods, “cleaving the aptamer by the Endonuclease V enzymatic activity to reduce or eliminate the binding of the oligonucleotide aptamer to the DNA polymerase and activate the DNA synthesis activity of the DNA polymerase” is preferably complete (100%) or substantially complete (e.g., inhibited to a level in the range of about 80% to 99.99%, or to any subrange or level therein), but can be partial (e.g., inhibited to a level in the range of from about 1% to about 99.99%), as exemplified herein (e.g., FIGS. 3, 5, 7, and 9).

An oligonucleotide aptamer may comprise ribo- or 2′-deoxyribonucleotides or a combination thereof. Oligonucleotide aptamers may be modified. Regarding the aptamers herein, the terms “modified” and “modification” are used in two different aspects, wherein the aptamers can be (i) modified synthetically, e.g. during the oligonucleotide synthesis, and (ii) enzymatically-modified in the context of or during DNA synthesis reactions. Synthetically, the aptamers may incorporate any kind and/or number of structural modifications across the length of the aptamer (e.g., in the middle or at the ends of the oligonucleotide chain). The term “structural modifications” refers to any chemical substances such as atoms, moieties, residues, polymers, linkers or nucleotide analogs, etc., which are usually of a synthetic nature and which are not commonly present in naturally-occurring nucleic acids. As used herein, the term “structural modifications” also include nucleoside or nucleotide analogs which are rarely present in naturally-occurring nucleic acids including but not limited to inosine, 5-bromouracil, 5-methylcytosine, 5-iodouracil, 2-aminoadenosine, 6-methyladenosine, pseudouridine, deoxyuridine, and the like. In particular embodiments, the aptamers incorporate one or more deoxyinosine (deoxyriboinosine) nucleotides that enable an endonuclease V enzyme to recognize the oligonucleotide aptamer as a substrate and modify its structure by cleavage. Nucleotides in the aptamers may be modified at the phosphates, sugar moieties or nucleotide bases. The structural modifications can be “duplex-stabilizing modifications.” “Duplex-stabilizing modifications” refer to structural modifications, the presence of which in double-stranded nucleic acids provides a duplex-stabilizing effect when compared in thermal stability, usually measured as “Tm,” with respective nucleic acid complexes that have no such structural modification and, e.g., comprise natural nucleotides. Duplex-stabilizing modifications are structural modifications that are most commonly applied in synthesis of probes and primers, as represented by modified nucleotides and ‘tails’ like intercalators and minor groove binders as, for example, disclosed in U.S. Pat. No. 8,349,556, issued to Kutyavin, I. V.; U.S. Pat. No. 7,794,945, issued to Hedgpeth, J., et al.; U.S. Pat. No. 6,127,121, issued to Meyer, Jr., R. B., et al.; U.S. Pat. No. 5,801,155, issued to Kutyavin, I. V., et al., and the references cited in. Duplex-stabilizing modifications can be used to prepare aptamers of the invention, for example, to improve thermal stability of stem (duplex) structures of hairpin-like aptamers. In preferred methods of the invention, the oligonucleotide aptamers are modified in (e.g., during) DNA synthesis reactions using enzymatic activity of one or more aptamer-modifying enzyme(s). In this aspect, the terms “modify,” “modification,” and “structural modifications” mean changes in the initial chemical structure of the aptamers. The change is triggered by Endonuclease V enzymes, resulting in a cleavage of one or more phosphodiester bonds. In the methods of the invention, these Endonuclease V-triggered structural modifications reduce or eliminate the ability of the oligonucleotide aptamer to bind to the DNA polymerase and block or reduce its activity in the reaction. In certain aspects, oligonucleotide aptamers comprise one or more deoxyinosine nucleotides, and aptamer modification results from aptamer cleavage (phosphodiester bond cleavage) by endonuclease V, at the second phosphodiester bond in the DNA strand on the 3′ side of a deoxyinosine nucleotide.

In particular aspects, the term “secondary structure” refers to an intramolecular complex formation of one sequence in a poly- or oligonucleotide with another sequence in the same polymer due to complete or partial complementarity between these two sequences formed based on the principal rules of Watson-Crick base pairing. The terms “hairpin” structure and “stem-loop” structure as referred to herein describe elements of secondary structure, and both terms refer to a double-helical region (stem) formed by base pairing between complementary sequences within a single strand RNA or DNA.

As used herein, the term “nuclease” refers to an enzyme that expresses a phosphomonoesterase or phosphodiesterase activity and is capable of cleaving a phosphoester bond in compounds such as R′—O—P(O)(OH)₂ and R′—O—P(O)(OH)—O—R,” resulting in products R′—OH+P(O)(OH)₃ and R′—OH+P(O)(OH)₂—O—R″ (or R″—OH+P(O)(OH)₂—O—R′), respectively, and wherein R′ and R″ may be moieties of any structure which are not necessarily of a nucleotide nature. The term “nucleases” incorporates both “exo” and “endo” nucleases. In certain aspects, endonuclease V is used to cleave phosphodiester bonds of oligonucleotide aptamers comprising one or more deoxyinosine nucleotides.

The term “aptamer-modifying enzymatic activity” refers to an aptamer-modifying enzyme or mixture of aptamer-modifying enzymes which recognize an aptamer of the invention as a substrate and modifies its structure so that the inhibitory activity of the aptamer is substantially disabled. In particular aspects, this recognition is directed by the presence of one or more deoxyinosine nucleotides within the aptamer structure. These aptamer structural modifications caused by endonuclease V enzymatic activity reduce the ability of the oligonucleotide aptamer to bind to the DNA polymerase and to block or reduce the polymerase activity in the reaction mixture.

As used herein, the term “deoxyinosine” refers to deoxyinosine and structural modifications of deoxyinosine, including but not limited to 7-deaza-deoxyinosine, 8-aza-7-deaza-deoxyinosine and other structural modifications of deoxyinosine, and including various ribonucleotide derivatives thereof, which can be recognized and cleaved by an endonuclease V enzymatic activity.

As used herein, the term “deoxyuridine” refers to deoxyuridine and structural modifications of deoxyuridine, including but not limited to various ribonucleotide derivatives thereof, that can be recognized and cleaved by an endonuclease V enzymatic activity.

As used herein, the term “endonuclease V” refers to repair enzymes that recognize deoxyinosine in DNA, and hydrolyze the second phosphodiester bond in the DNA strand on the 3′ side of a deoxyinosine nucleotide (see, e.g., U.S. Pat. No. 8,143,006, issued to Kutyavin, I. V.).

The term “DNA polymerase” refers to an enzyme that catalyzes synthesis of deoxyribo nucleic acids (DNAs), most commonly double-stranded DNAs, using single-stranded DNAs as “templates.” The DNA synthesis is usually initiated by an oligonucleotide primer that is hybridized to a complementary template strand. Starting from this template-hybridized primer, DNA polymerase creates a Watson-Crick complementary strand in the presence of 2′-deoxyribonucleotide 5′-triphosphates (dNTPs). The term “DNA polymerase,” as used herein, also incorporates “reverse transcriptases,” enzymes which can perform DNA synthesis using single-stranded ribonucleic acids (RNAs) as template strands.

“Polynucleotide” and “oligonucleotide” are used herein interchangeably and in each case means a linear polymer of nucleotide monomers. Polynucleotides typically range in size from a few monomeric units, e.g., 5-60, when they are usually referred to as “oligonucleotides,” to several thousand monomeric units. The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. The oligonucleotides may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof. Unless otherwise specified, whenever a polynucleotide or oligonucleotide is represented by a sequence of letters, for example, “TTCTTAGCGTTT (SEQ ID NO:18),” it is understood herein, unless otherwise specified in the text, that the nucleotides are in 5′→3′ order from left to right and that “A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotes deoxythymidine. Usually DNA polynucleotides comprise these four deoxyribonucleosides linked by phosphodiester linkage whereas RNA comprises uridine (“U”) in place of “T” for the ribose counterparts.

The terms “oligonucleotide primer” and/or “primer” refer to a single-stranded DNA or RNA molecule that hybridizes to a complementary target nucleic acid and serves to prime enzymatic synthesis of a second nucleic acid strand in the presence of a DNA polymerase. In this case, the target nucleic acid “serves as a template” for the oligonucleotide primer.

In particular aspects, the terms “complementary” or “complementarity” are used herein in reference to the polynucleotide base-pairing rules. Double-stranded DNA, for example, consists of base pairs wherein, for example, G complexes or pairs with C via formation of a three hydrogen bond complex, and A complexes or pairs with T via formation of a two hydrogen bond complex, such that G is regarded as being complementary to C, and A is regarded as being complementary to T. In this sense, for example, an oligonucleotide 5′-GATTTC-3′ is complementary to the sequence 3′-CTAAAG-5′ via intrastrand G:C and A:T hydrogen bonding interactions. Complementarity may be “partial” or “complete.” In partial complementarity, only some of the nucleic acids bases are matched according to the base pairing rules. “Complementarity” may also be used in reference to individual nucleotides and oligonucleotide sequences within the context of polynucleotides (e.g., inter-strand complementarity). The terms “complementary” and “complementarity” refer to the most common type of complementarity in nucleic acids, namely, Watson-Crick base pairing as described above, although the oligonucleotides may alternately participate in other types of “non-canonical” pairings like Hoogsteen, wobble and G-T mismatch pairing.

The term “natural nucleosides” refers to the four standard 2′-deoxyribonucleosides (usually named herein as “deoxynucleosides” or “deoxyribonucleosides”) that are found in DNAs isolated from natural sources. Natural nucleosides are deoxyadenosine, deoxycytidine, deoxyguanosine, and deoxythymidine. The term also encompasses their ribose counterparts, with uridine (U) in place of thymidine. The same name variations are applied herein to “natural nucleotides.”

As used herein, the terms “unnatural nucleosides” or “modified nucleosides” refer to nucleoside analogs that are different in their structure from those natural nucleosides for DNA and RNA polymers. Some naturally occurring nucleic acids contain nucleosides that are structurally different from the natural nucleosides defined above, for example, DNAs of eukaryotes may incorporate 5-methyl-cytosine, and tRNAs contain many nucleoside analogs. However, as used herein, the terms “unnatural nucleosides” or “modified nucleosides” encompasses these nucleoside modifications even though they can be found in natural sources.

The term “reaction mixture” generally means herein a solution containing all necessary reactants for performing DNA synthesis such as a DNA polymerase, oligonucleotide primer(s), template polynucleotide, deoxyribonucleoside 5′-triphosphates, reaction cofactors (e.g., magnesium or manganese ions), etc. The reaction mixture can incorporate other reaction components that help to improve DNA synthesis (e.g., buffering and salt components, detergents, proteins like bovine serum albumin (BSA), scavengers, etc.) or components that are necessary for detection of the newly synthesized DNA molecules such as, for example, fluorescent dyes and oligonucleotide probes. A reaction mixture is usually prepared at low temperatures at which enzymatic components are inactive, for example, by mixing the components on ice at ˜0° C. When the reactions are ready, the mixtures can be heated to the desired reaction temperatures. In this aspect, the term “reaction temperature” refers to a temperature or a temperature range at which DNA synthesis is performed. In case of PCR reactions, it is usually taken as the lowest thermo-cycling temperature, commonly called the annealing temperature.

“dNTPs” is an abbreviation of a mixture of two or more of the four natural deoxynucleoside 5′-triphosphates that are useful to facilitate primer extension with a DNA polymerase and/or amplification. Respectively, the abbreviations “dATP,” “dCTP,” “dGTP,” and “dTTP” correspond to the individual nucleotides. In some embodiments, the four dNTPs are present at equal concentrations. In other embodiments, the concentrations of the dNTPs are not all identical. In some embodiments, fewer than all four dNTPs are present. For example, only one dNTP may be present, or a pair-wise combinations, or three of four dNTPs may be present in the mixture.

In some aspects, “amplification” and “amplifying” deoxyribonucleic acids, in general, refer to a procedure wherein multiple copies of DNA of interest are generated. The DNA amplification can be performed at a constant temperature using “isothermal amplification reactions.” Examples of isothermal amplification reactions include, but are not limited to, Strand Displacement Amplification (SDA) (U.S. Pat. No. 5,270,184 to Walker, G. T., et al.; U.S. Pat. No. 6,214,587 to Dattagupta, N., et al.), Rolling Circle amplification (RCA) (U.S. Pat. No. 5,854,033 to Lizardi, P.), Loop-Mediated Amplification (LMA) (U.S. Pat. No. 6,410,278 to Notomi, T., and Hase, T.), isothermal amplification using chimeric or composite RNA/DNA primers (U.S. Pat. No. 5,824,517 to Cleuziat, P., and Mandrand, B.; U.S. Pat. No. 6,251,639 to Kurn, N.), Nucleic Acid Sequence-Based Amplification (NASBA) (U.S. Pat. No. 6,063,603 to Davey, C. and Malek, L. T.), and many other methods.

“PCR” is an abbreviation of “polymerase chain reaction,” an art-recognized nucleic acid amplification technology (e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis, K. B.). Commonly used PCR protocol employs two oligonucleotide primers, one for each strand, designed such that extension of one primer provides a template for the other primer in the next PCR cycle. Generally, a PCR reaction consists of repetitions (cycles) of (i) a denaturation step that separates the strands of a double-stranded nucleic acid, followed by (ii) an annealing step, which allows primers to anneal to positions flanking a sequence of interest on a separated strand, and then (iii) an extension step that extends the primers in a 5′ to 3′ direction, thereby forming a nucleic acid fragment complementary to the target sequence. Each of the above steps may be conducted at a different temperature using an automated thermocycler. The PCR cycles can be repeated as often as desired resulting in an exponential accumulation of a target DNA fragment whose termini are usually defined by the 5′-ends of the primers used. Although conditions of PCR can vary in a broad range, a double-stranded target nucleic acid is usually denatured at a temperature of >90° C., primers are annealed at a temperature in the range of about 50-75° C., and the extension is preferably performed in a 72-75° C. temperature range. In PCR methods, the annealing and extension can be combined into one stage (i.e., using a single temperature). The term “PCR” encompasses its numerous derivatives such as “RT-PCR,” “real-time PCR,” “nested PCR,” “quantitative PCR,” “multiplexed PCR,” “asymmetric PCR,” and the like.

“Real-time detection” means an amplification reaction for which the amount of reaction product, i.e., target nucleic acid, is monitored as the reaction proceeds. Real-time detection is possible when all detection components are available during the amplification and the reaction composition and conditions support both stages of the reaction, the amplification and the detection.

As used herein, the term “kit” refers to any system for delivering materials. In the context of reaction assays, such delivery systems include elements allowing the storage, transport, or delivery of reaction components such as oligonucleotides, buffering components, additives, reaction enhancers, enzymes, and the like in the appropriate containers from one location to another commonly provided with written instructions for performing the assay. Kits may include one or more enclosures or boxes containing the relevant reaction reagents and supporting materials. The kit may comprise two or more separate containers wherein each of those containers includes a portion of the total kit components. The containers may be delivered to the intended recipient together or separately.

In general, the term “design” in the context of the methods has broad meaning and in certain respects is equivalent to the term “selection.” For example, the terms “primer design” and “aptamer design” can mean or encompass selection of a particular oligonucleotide structure including the nucleotide primary sequence and structural modifications (e.g., labels, modified nucleotides, linkers, etc.). In particular aspects, the terms “system design” and “assay design” relate to the selection of any, sometimes not necessarily to a particular, methods including all reaction conditions (e.g., temperature, salt, pH, enzymes, including the aptamer-modifying enzymes and DNA polymerase, oligonucleotide component concentrations, etc.), structural parameters (e.g., length and position of primers and probes, design of specialty sequences, etc.) and assay derivative forms (e.g., post-amplification, real time, immobilized, FRET detection schemes, etc.) chosen to amplify and/or to detect the nucleic acids of interest.

Reversible Blocking of DNA Synthesis Activity of DNA Polymerases Using Circular Oligonucleotide Aptamers.

Prior use of oligonucleotide aptamers during DNA synthesis have attempted to block DNA polymerase activity, preferably completely, at low temperatures, while releasing (activating) the DNA polymerase activity, preferably completely, at an elevated reaction temperature. It is difficult, however, to achieve complete ‘block-and-release’ formats using conventional aptamer-based methods (e.g., Yakimovich, O. Yu., et al. (2003); Jayasena, S. D. (1999); U.S. Pat. No. 5,693,502 to Gold, L., and Jayasena, S. D. (1997)). Effective blockage of DNA polymerase at low temperatures commonly leads to ineffective release of the enzyme at the elevated reaction temperature and vice versa. Aspects of the present invention provide a solution to this long-standing problem in the art. As in the conventional approaches cited above, the DNA polymerase activity is blocked or reduced in methods of the invention by the presence of a circular oligonucleotide aptamer that binds to the DNA polymerase, blocking the DNA synthesis activity of the DNA polymerase. Unlike prior art techniques, however, in methods of the invention, the aptamer-inactivated DNA polymerase is activated by providing to a DNA synthesis reaction mixture an endonuclease V enzyme activity that recognizes the circular oligonucleotide aptamer as a substrate and cleaves its structure. This cleavage reduces or eliminates the binding of the circular oligonucleotide aptamer to the DNA polymerase and thereby reactivates the DNA synthesis activity of the DNA polymerase.

In some embodiments of the invention, activation of an aptamer-inactivated DNA polymerase in a reaction mixture, comprising (i) a DNA polymerase, (ii) circular oligonucleotide aptamer in an amount effective to inhibit the DNA synthesis activity of the DNA polymerase, (iii) an endonuclease V aptamer-modifying enzyme and other components necessary for DNA synthesis, is facilitated using a reaction temperature that accelerates (or facilitates) both DNA polymerase and endonuclease V enzymatic activities. For example, the reaction mixture can be prepared at low temperature (first temperature) at which a DNA polymerase is effectively blocked by an aptamer and an aptamer-modifying enzyme, endonuclease V, has sufficiently reduced or preferably no activity (e.g., at 0° C.), and then the activation of the aptamer-inactivated DNA polymerase is facilitated by heating the reaction to a temperature (second temperature) that accelerates or facilitates the aptamer-modifying endonuclease V enzymatic activity. If necessary, a DNA polymerase can be activated by the endonuclease V enzyme(s) at any temperature below the reaction temperature for DNA synthesis. This can be applied, for example, when a particular endonuclease V enzyme is unstable at the reaction temperature for DNA synthesis, for example, due to denaturation. In this case, the DNA polymerase is first activated at an intermediate temperature wherein the endonuclease V is active and then heated to the reaction temperature to perform DNA synthesis.

In some aspects, the reaction mixture is created by addition of aqueous solution to one or more reaction components which are initially in a dried state as disclosed, for example, in U.S. Pat. No. 3,721,725 to Briggs, A. R., and Maxwell, T. J. (1973) (incorporated herein by reference). For example, in some methods of the invention for DNA amplification and detection, the aqueous solution can be a sample solution or solution that contains one or more polynucleotide templates for DNA synthesis whereas all other reaction components such as the DNA polymerase, aptamer, endonuclease V enzyme, dNTPs, catalytic cofactors such as magnesium (Mg2+) and/or manganese (Mn2+) ion (e.g., provided as a chloride salt), buffering components, detergents, proteins like bovine serum albumin (BSA), scavengers, etc., are initially provided in a dry state.

In some aspects, DNA synthesis results in DNA amplification in the reaction mixture. The DNA amplification can be an isothermal amplification reaction, for example, as described in U.S. Pat. No. 5,270,184 to Walker, G. T., et al.; U.S. Pat. No. 6,214,587 to Dattagupta, N., et al.; U.S. Pat. No. 5,854,033 to Lizardi, P.; U.S. Pat. No. 6,410,278 to Notomi, T., and Hase, T.; U.S. Pat. No. 5,824,517 to Cleuziat, P., and Mandrand, B.; U.S. Pat. No. 6,251,639 to Kurn, N.; U.S. Pat. No. 6,063,603 to Davey, C., and Malek, L. T., and many other methods. In other aspects, the DNA amplification can be a PCR reaction (e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis, K. B.). In methods of the invention, the DNA amplification is performed for detection as well as measuring an amount of a target DNA in the reaction mixture.

As has been established in the art (e.g., Yao, M., Kow, Y. W., 1997), a number of nucleotide modifications can be recognized by the endonuclease V enzymes to provide for the oligonucleotide cleavage. For example, an oligonucleotide aptamer that contains one or more deoxyuridine nucleotide(s) as described, for example, in Yao, M., Kow, Y. W., 1997, can be used in methods, compositions and kits of the present invention, such that contact of such aptamers with an endonuclease V enzymatic activity causes cleavage of the deoxyuridine-containing aptamer. However, the preferred modified nucleotide in aptamers is one or more deoxyinosine nucleotide(s). The endonuclease V enzymes of the present invention can cleave single-stranded and double-stranded oligonucleotides. Therefore the circular aptamers of the present invention can be both, single and double-stranded. In particular embodiments, the circular aptamers of the invention are stem-loop or hairpin-like molecules. Effective use of these two exemplary combinations (endonuclease V)+(deoxyinosine-incorporating circular aptamers) is illustrated in the Examples provided herein using, in particular, hairpin-like structures (FIGS. 2-6; as described in working Example 3 herein below). Deoxyinosine nucleotide can be located either in loop or stem fragments of the hairpin-like circular aptamers of the invention. Preferred locations of deoxyinosine modifications in circular aptamers of the present invention are described herein.

The oligonucleotide aptamers as well as oligonucleotide primers and probes can be prepared by any method of oligonucleotide synthesis selected by a person of ordinary skill in the art, such as methods that utilize (2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidites (Example 1). Protected nucleotides and derivatives, linkers, dyes, tails, solid supports, and other appropriate components can be prepared by methods of organic chemistry or obtained from market providers such as, for example, Glen Research and Biosearch Technologies. Suppliers such as Integrated DNA Technologies and Biosearch Technologies also offer oligonucleotide custom synthesis including numerous structural modifications such as deoxyinosine and deoxyuridine. In the methods of the invention, selection of an aptamer structure, including one or more deoxyinosine nucleotides, is intended to achieve (i) complete deactivation of the DNA polymerase at the initial reaction assembly temperature, (ii) complete or substantially complete (or as much as possible) deactivation of the DNA polymerase at the elevated reaction temperature for DNA synthesis, and (iii) substantially complete or complete (or as much as possible) reactivation of this enzyme at the DNA synthesis reaction temperature once the circular aptamer has been modified by the endonuclease V enzymatic activity. Generally, endonuclease V enzymes do not interfere with DNA synthesis (primer extension) or DNA amplification. The location and number of deoxyinosine nucleotides within a circular aptamer, as well as the rate and efficiency of the endonuclease V enzymes is preferably taken into consideration. Preference is given to deoxyinosine locations within a circular aptamer that have little or no negative effect on stability of the aptamer-DNA polymerase complex, but sufficiently disturb the structural integrity of the circular aptamer so that the cleaved aptamer does not bind to or inhibit the DNA polymerase. Results from circular aptamers containing deoxyinosine nucleotides (SEQ ID NOS:7-15 and 12-20) as the endonuclease V-recognition motif are illustrated in FIGS. 3, 5, and 7. Surprisingly, even a single base modification at numerous locations showed excellent results in the exemplary assays.

According to the prior art (Yakimovich, O. Yu., et al. (2003); Jayasena, S. D. (1999); U.S. Pat. No. 5,693,502 to Gold, L. and Jayasena, S. D.), efficiency of hairpin-type aptamer binding to DNA polymerase is determined by (i) a loop segment, which is a substantially conservative sequence, and (ii) length of the stem duplex, which preferably needs to be ˜19-20 base pairs or longer. In addition, the present inventors have found that in addition to the sequence, the circularization of the hairpin-like aptamers can be another important factor affecting the stability of an aptamer-polymerase complex. For example, the stem sequence of aptamer SEQ ID NO:5 (FIG. 2) in the present working Examples mostly comprises A and T nucleotides. As illustrated in FIGS. 3A and 3B, this aptamer SEQ ID NO:5 has little, if any DNA-polymerase deactivating affect during PCR, whereas it circular analog SEQ ID NO:6 was very effective in DNA-polymerase deactivation (FIGS. 3 and 5). Moreover, the deoxyinosine-incorporating circular analog SEQ ID NO:12 was very effective in blocking not only Taq (FIGS. 5A and 5B), but also many other DNA polymerases (FIG. 7). Out of six DNA polymerases investigated, only Bst polymerase (large fragment) was not inactivated by the circular aptamer SEQ ID NO:12. Analysis of FIG. 7 points to additional surprising results. First, regardless of the difference in reaction temperature, circular aptamer SEQ ID NO:12 blocked Phusion® polymerase much more efficiently at 65° C. than Taq polymerase at 60° C. According to additional surprising aspects of the invention, therefore, the polymerase-blocking efficiency of the circular aptamer SEQ ID NO:12 varies for the DNA polymerases investigated. Second, the duplex sequence in an aptamer of the invention can be further optimized by base pair changes for even better polymerase blockage in each particular case. Third, using the sequence of circular aptamer SEQ ID NO:6 as an origin, sequence optimization for strongest binding can be performed for every DNA polymerase known in the art, although for some DNA polymerases like Bst, optimization may require alterations in the loop segment of the aptamer as well as in the stem. In this sense, the present disclosure also provides methods of screening for more optimal aptamers for use in the disclosed methods.

Circular aptamers of the invention, whether single-stranded or double-stranded, can contain any number of modified nucleotides, internal linkers and moieties, and other structural modifications as long as these modifications do not interfere with the DNA polymerase deactivation and subsequent reactivation processes during DNA synthesis. Preference should be given to structural modifications that help to deactivate the DNA polymerase and do not adversely affect the endonuclease V enzyme activation reaction. Both loop and stem fragments can be modified in the circular hairpin-type aptamers. Although the loop segments described in Yakimovich, O. Yu., et al. (2003), Jayasena, S. D. (1999), and U.S. Pat. No. 5,693,502 to Gold, L. and Jayasena, S. D., contain conserved sequence motifs, the results in FIG. 5 (Example 3) herein show that deoxyinosine substitution(s) at certain loop positions have very little (SEQ ID NO:14) or no effect (SEQ ID NO:12) on aptamer performance (FIG. 5). The circular hairpin-like aptamers used in the working Examples of the present invention (structures shown in FIGS. 2 and 4) were designed to incorporate one short 5′-GAA-3′ loop (second loop) on the end of the double-stranded stem opposite the end having the conservative 5′-TTCTTAGCGTTT-3′ (SEQ ID NO:18) loop sequence. In methods of the invention, this second loop can vary in length and nucleotide sequence and composition. It can be, for example, the same conservative sequence 5′-TTCTTAGCGTTT-3′ (SEQ ID NO:18), or a different sequence and/or different length, etc.

Methods of the invention can be performed using one or more reaction temperatures wherein a DNA polymerase and endonuclease V express suitable activity. Specificity of DNA synthesis is usually increased at higher temperatures, and therefore thermostable enzymes are usually preferred. The upper level of the reaction temperature can be selected based on the DNA polymerase stability. In cases when an endonuclease V is not stable at a desired reaction temperature, the DNA polymerase activation can be initiated at a lower intermediate temperature wherein the endonuclease V is stable and active and then raised to the desired reaction temperature for DNA synthesis (primer extension). In some embodiments, a DNA polymerase is preferably first deactivated by contacting (e.g., by combining or mixing) with an aptamer before other reaction components of the DNA synthesis are added. Molar reaction concentration of an aptamer applied should be at least equal to the concentration of a DNA polymerase or preferably greater. Market providers commonly do not disclose the molar amount of the enzymes, therefore the precise excess of the aptamers over the DNA polymerases used in Examples provided herein was not known. However, the aptamers used in the Examples below were estimated to be present in a range of ˜40-80 fold, or greater, molar excess relative to DNA polymerase. In some embodiments, the aptamer is present in a molar excess (ratio) over the DNA polymerases of at least ˜5-fold, although the ratio can be higher or lower than 5-fold. The amounts of the enzymes, circular aptamers and other reaction components used in the reaction may be optimized and depend on many factors including, but not limited to selection of the particular enzymes, enzymatic activities at the reaction temperature, reaction temperature itself, nature of the circular aptamers, and their special endonuclease V recognition motifs (e.g., deoxyinosine or deoxyuridine, etc.) to provide for cleavage of the aptamers. Methods of the present invention can be particularly useful for so-called ‘fast’ PCR with a cycle time shorter than 20 seconds.

In certain embodiments, methods of the invention can be practiced using a kit comprising a DNA polymerase-binding circular oligonucleotide aptamer recognizable and modifiable by an endonuclease V enzymatic activity, and an endonuclease V enzyme activity to provide for specific cleavage of the circular aptamer. The kit can also include a DNA polymerase that is initially deactivated by the circular oligonucleotide aptamer. Alternatively, the kit can include, in addition to the endonuclease V enzyme activity, a complex of the DNA polymerase with the circular oligonucleotide aptamer, wherein the components of this complex are present at a specific and optimal molar ratio. As a matter of convenience, such a kit can include components allowing the storage, transport and other reaction components such as oligonucleotides, buffering components, additives, reaction enhancers, etc. The circular aptamers of the kits can be single-stranded or have a stem-loop structure, and they can incorporate one or more special structural features. The kits can be used for the DNA synthesis, amplification as well as the detection of the amplified DNA fragments.

In some embodiments, the invention includes a reaction mixture comprising a DNA polymerase, a circular oligonucleotide aptamer that binds to the DNA polymerase and present in an amount effective to inhibit DNA synthesis activity of the DNA polymerase, an endonuclease V enzyme activity that is capable of cleaving the oligonucleotide aptamer to reduce or eliminate binding of the oligonucleotide aptamer to the DNA polymerase, and other reaction components necessary for DNA synthesis is also a subject of the present invention. In some embodiments, the reaction mixture can be assembled using concentrated stock solutions of one or more components, usually in water to provide the desired component concentration in the final mixture. Mixing is recommended to be performed at a low temperature (e.g., close to 0° C.) at which the enzymes, particularly endonuclease V enzymes, are inactive. Preferably, the reaction mixture should be used for DNA synthesis soon after preparation. Storage of a fully assembled reaction mixture is not recommended. However, reaction components including enzymes can retain activity for a long time (days, months or even years) in a dried state. For example, in some embodiments of the invention, one or more of the components for forming a mixture of the invention can be provided in a dried form, such as dried beads as described, for example, in U.S. Pat. No. 3,721,725 to Briggs, A. R., and Maxwell, T. J. (1973), including (but not limited to) DNA polymerase, circular oligonucleotide aptamer, and endonuclease V enzyme activity such that one or more of the components is prepared in a form of dried beads as described, for example, in U.S. Pat. No. 3,721,725 to Briggs, A. R., and Maxwell, T. J. (1973). In some embodiments, the mixture comprises DNA polymerase, a circular oligonucleotide aptamer that binds to the DNA polymerase and present in an amount effective to inhibit DNA synthesis activity of the DNA polymerase, and an endonuclease V enzyme activity that is capable of cleaving the oligonucleotide aptamer to reduce or eliminate binding of the circular oligonucleotide aptamer to the DNA polymerase, which are mixed together in a dried form.

Example 1 Synthesis of 5′-Phosphate-Incorporating Oligonucleotides, Noncircular Aptamer, Primers and Fluorescent Probes

Standard phosphoramidites, including modified nucleotide analogs such as deoxyinosine phosphoramidite (Catalog Number: 10-1040-xx), a phosphoramidite for incorporation of 5′-phosphate moiety, solid supports and reagents to perform solid support oligonucleotide synthesis, were purchased from Glen Research. A 0.25 M 5-ethylthio-1H-tetrazole solution was used as a coupling agent. Oligonucleotides were synthesized either on ABI394 DNA synthesizer (Applied Biosystems) or MerMaid 6 DNA synthesizer (BioAutomation Corporation) using protocols recommended by the manufacturers for 0.2 μmole synthesis scales. Fluorescein (FAM) conjugated to 5-position of deoxyribouridine (U) of probe SEQ ID NO:29 (FIG. 8) was introduced to the hairpin during oligonucleotide synthesis using 5′-dimethoxytrityloxy-5-[N-((3′,6′-dipivaloyl fluoresceinyl)-aminohexyl)-3-acrylimido]-2′-deoxyribouridine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Glen Research, Catalog Number: 10-1056-xx). A 6-fluorescein reporting dye was incorporated onto the 5′-end, and a BHQ1 quencher was introduced to the 3′-end of probe SEQ ID NO:3 (FIG. 1) using respective phosphoramidite and CPG from Biosearch Technologies (Catalog numbers: BNS-5025 and BG1-5041G). After the automated synthesis, oligonucleotides were deprotected in aqueous 30% ammonia solution by incubation for 12 hours at 55° C. or 2 hours at 70° C.

Tri-ON oligonucleotides were purified by HPLC on a reverse phase C18 column (LUNA 5 μm, 100 A, 250×4.6 mm, Phenomenex Inc.) using gradient of acetonitrile in 0.1 M triethyl ammonium acetate (pH 8.0) or carbonate (pH 8.5) buffer with flow rate of 1 ml/min. A gradient profile including washing stage 0-14% (10 sec), 14→45% (23 min), 45→90% (10 min), 90→90% (5 min), 90→0% (30 sec), 0→0% (7 min) was applied for purification of all Tri-ON oligonucleotides. The product-containing fractions were dried down in vacuum (SPD 1010 SpeedVac, TermoSavant) and trityl groups were removed by treatment in 80% aqueous acetic acid at room temperature for 40-60 min. After addition to the detritylation reaction (100 μl) of 20 μl sodium acetate (3 M), the oligonucleotide components were precipitated in alcohol (1.5 ml), centrifuged, washed with alcohol and dried down. Concentration of the oligonucleotide components was determined based on the optical density at 260 nm and the extinction coefficients calculated for individual oligonucleotides using on-line OligoAnalyzer 3.0 software provided by Integrated DNA Technologies. Based on the measurements, convenient stock solutions in water were prepared and stored at −20° C. for further use. The purity of all prepared oligonucleotide components was confirmed by analytical 8-20% PAAG electrophoresis, reverse phase HPLC and by spectroscopy on Cary 4000 UV-VIS spectrophotometer equipped with Cary WinUV software, Bio Package 3.0 (Varian, Inc.).

Example 2 Synthesis of Circular Oligonucleotide Aptamers

Exemplary circular stem-loop aptamers SEQ ID NOS:6-15 were prepared by ligation of the corresponding 5′-phosphate incorporating oligonucleotides shown in FIGS. 2 and 4 using T4 DNA Ligase kit from New England Biolabs (Catalog number: M0202M). The reaction mixtures were prepared by mixing 67 μl of 10× ligation buffer and 2.5 μl of T4 DNA ligase (2,000 U/μl) from the kit with 10 optical units (at 260 nm) of a 5′-phosphate-labelled oligonucleotide (FIGS. 2 and 4) and deionized water to provide 670 μl of the final reaction volume. The reaction mixtures were left at room temperature for 1 hour and then heated to 65° C. for 15 min. According to HPLC analysis, the ligation reactions were nearly quantitative (˜95%), and the circular aptamers were isolated by HPLC chromatography as described in Example 1. The collected fractions were dried down, and the circular aptamers were dissolved in water. Concentration of the circular aptamers was determined based on the optical density at 260 nm as also described in Example 1. Based on the measurements, convenient stock solutions in water were prepared and stored at −20° C. for further use.

Example 3 Application of Deoxyinosine-Containing Circular Aptamers to Control Polymerase Activity of Taq Polymerase

This working example shows application of deoxyinosine-containing circular hairpin-like aptamers to control activity of Taq polymerase during PCR.

For the results shown in FIGS. 3 and 5, reaction mixtures (25 μL) were prepared on ice by mixing corresponding stock solutions to provide 200 nM forward primer (FIG. 1, SEQ ID NO:1), 300 nM reverse primer (SEQ ID NO:2), 200 nM FRET probe (SEQ ID NO:3), 0.02 U/μL Taq DNA polymerase (GenScript cat no: E00007), dNTPs (200 μM each), bovine serum albumin (0.1 μg/μL), 100 ng of human genomic DNA (GenScript cat no: M00094) and, when present, one of the aptamers SEQ ID NOS:5-15 (80 nM) in 5 mM MgCl₂, 50 mM KCl, 20 mM Tris-HCl (pH8.0). The reaction tubes were quickly transferred into SmartCycler instrument (Cepheid Corporation) and temperature cycling initiated. The PCR time/temperature profile comprised initial incubation at 95° C. for 15 seconds followed by 50 cycles of incubation at 95° C. for 1 second and then at 60° C. for 20 seconds. The reaction fluorescence was measured in every PCR cycle during the annealing/extension stage (60° C.) and the results are shown in FIGS. 3 and 5. Each fluorescence curve is an average of four identical reactions. Initial background fluorescence was subtracted by the instrument software.

The reaction conditions used to generate the fluorescence profiles shown in FIGS. 3 and 5 were identical except for the presence or absence of endonuclease V enzymatic activity and the presence or absence of different oligonucleotides as potential inhibitors of polymerase activity. PCR reactions were performed either in the absence (left diagram of each figure) or presence (right diagram) of T. maritima endonuclease V (0.04 U/μL, Fisher Scientific cat no: FEREN0141) in the reaction mixtures. Structures of the oligonucleotide aptamers used in the experiments of FIGS. 3 and 5 are shown in FIGS. 2 and 4, respectively.

In summary of this working Example, the real-time curves shown in FIG. 3 show that unmodified noncircular aptamer SEQ ID NO:5 has very little, if any polymerase-deactivating capability whereas its circular analog SEQ ID NO:6 effectively blocks Taq polymerase activity during PCR, which is not affected by the presence of endonuclease V activity in the reaction mixture. This result illustrates the effectiveness of aptamer circularization for the deactivation of Taq DNA polymerase. Analysis of the real-time PCR curves in FIG. 3 leads to the following conclusions. Virtually identical and very effective results of DNA polymerase inactivation-activation were obtained for the aptamers SEQ ID NOS:7 and 8 incorporating deoxyinosine in the 5′-3′ duplex strand (the strand assignment is shown on the circular aptamer SEQ ID NO:6 of FIG. 2), whereas the results of the deoxyinosine incorporation into the 3′-5′ duplex strand of aptamers SEQ ID NOS:9-11 varied significantly. For example, circular aptamer SEQ ID NO:9 was very effective at DNA polymerase deactivation (FIG. 3A), but appeared to be refractory to DNA polymerase activation by endonuclease V (FIG. 3B). A likely explanation of these results is that the stem cleavage product of this circular aptamer (SEQ ID NO:9) functions as a self-priming hairpin; that is, the cleaved stem functions like the self-priming fluorescent probe (SEQ ID NO:16 of FIG. 6) used herein in the exemplary assays to detect and measure DNA polymerase activity (FIGS. 7A through 7F). Although the cleaved stem of the circular aptamer (SEQ ID NO:9) is very A-T rich, it is 10 base pairs long and therefore would be relatively stable at 60° C. Extension of this stem cleavage product by DNA polymerase likely competes against targeted amplification of the β2-microglobulin sequence (FIG. 1) by the DNA polymerase, resulting in apparent, but not actual, refractory reactivation due to an effective reduction in the rate of targeted amplification by the DNA polymerase, rather than the circular aptamer SEQ ID NO:9 being refractory to cleavage by endonuclease V. Positioning deoxyinosine closer to the 5′-GAA-3′ loop in circular aptamers SEQ ID NOS:10 and 11 destabilized the stem of the cleavage product, eliminating the self-priming problem. Activity of the DNA polymerase, initially blocked by these aptamers, was completely restored in the presence of endonuclease V (FIG. 3B), but these aptamers were less effective in initial DNA polymerase inactivation (FIG. 3A), with circular aptamer SEQ ID NO:10 performing the worst in this regard. In conclusion, preferred locations of deoxyinosine substitutions within the stem segment identified herein for highly homologous circular hairpin-like aptamers SEQ ID NOS:6-15 can be design-specific and may change depending on the stem length, base pair composition (e.g., G-C richness of duplexes) and particular sequences, etc.

Regarding ability of the circular hairpin-like aptamers to inactivate Taq DNA polymerase, the loop sequence 5′TTCTTAGCGTTT3′ (SEQ ID NO:18) is known to be highly conserved (e.g., Yakimovich, O. Yu., et al. (2003), Jayasena, S. D. (1999), and U.S. Pat. No. 5,693,502 to Gold, L., and Jayasena, S. D.). Nevertheless, a number of the nucleotide substitutions by deoxyinosine in the loop of circular aptamers SEQ ID NOS:12-15 shown in FIG. 4 were investigated, and the real-time PCR results are provided in FIG. 5. Significant differences in properties were found among these four aptamers. For example, the circular aptamer SEQ ID NO:15 was very ineffective in deactivation of Taq DNA polymerase. In contrast, the circular aptamer SEQ ID NO:14 was highly effective for polymerase deactivation (FIG. 5A), but failed to provide complete Endonuclease V-mediated reactivation (FIG. 5B); that is, circular aptamer SEQ ID NO:14 provided only partial enzyme reactivation. While not being bound by mechanism, this observation could reflect a reduced rate of cleavage by Endonuclease V at that particular deoxyinosine location within the loop, perhaps due to loop structural constraints. The aptamers SEQ ID NOS:14 and 15 represent a homologous guanine-to-hypoxanthine purine substitution. The most surprising results were obtained for the circular aptamers SEQ ID NOS:12 and 13 comprising nonhomologous (pyrimidine-purine) thymine-hypoxanthine base alterations within the highly conservative loop sequence. Both aptamers inactivated the DNA polymerase (FIG. 5A), although the aptamer SEQ ID NO:13 was a much less effective inhibitor than the aptamer SEQ ID NO:12, which was very effective in both deactivation (FIG. 5A) and the Endonuclease V-mediated activation (FIG. 5B) tests. In conclusion, investigation of the deoxyinosine substitutions within the aptamer loop sequences provided for identification of at least three exemplary nucleotide locations that can be used in embodiments of the present invention. For example, the loop sequences 5′-TTCITAGCGTTT-3′ (SEQ ID NO:19), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20), 5′-TTCTTAICGTTT-3′ (SEQ ID NO:21), 5′-TTCIIAGCGTTT-3′ (SEQ ID NO:22), 5′-TTCITAICGTTT-3′ (SEQ ID NO:23), 5′-TTCTIAICGTTT-3′ (SEQ ID NO:24), and 5′-TTCIITAICGTTT-3′ (SEQ ID NO:25) incorporating 1, 2, or 3 deoxyinosine nucleotides can be used in design of the circular hairpin-like aptamers for the methods of the present invention.

Example 4 Kinetics of Activation by Endonuclease V of Various DNA Polymerases Initially Blocked by Deoxyinosine-Containing Aptamer

This working example shows the kinetics of activation by endonuclease V of Taq (GenScript cat no: E00007), Q5® (New England Biolabs cat no: M0491S), Vent® (New England Biolabs cat no: M0254S), Deep Vent® (New England Biolabs cat no: M0258S), Bst large fragment (New England Biolabs cat no: M0275S), and Phusion® (New England Biolabs cat no: M0530S) DNA polymerases initially blocked by a deoxyinosine-containing aptamers.

For FIG. 7, reaction mixtures (25 μL) were prepared on ice by mixing corresponding stock solutions to provide self-priming hairpin SEQ ID NO:16 (200 nM, FIG. 8), a DNA polymerase (0.008 U/μL), dNTPs (200 μM each), bovine serum albumin (0.1 μg/μL) and, when present, the aptamer SEQ ID NO:12 (80 nM, FIG. 4) and T. maritima endonuclease V (0.02 U/μL, Fisher Scientific cat no: FEREN0141) in 5 mM MgCl₂, 50 mM KCl, 20 mM Tris-HCl (pH8.0). During preparation of the reaction mixture, the self-priming hairpin (SEQ ID NO:16) and endonuclease V were always added last to a premixed solution. Then the reaction tubes were transferred into a SmartCycler instrument (Cepheid Corporation) and heated to 60 or 65° C. as indicated for each fluorescence profile in FIG. 7. The reaction fluorescence was monitored every 7 seconds. The plotted curves are the averages of four paralleled identical reactions. Initial background fluorescence was subtracted.

The results of FIG. 7 show that not only Taq polymerase, but also many other DNA polymerases can be inactivated and then activated using endonuclease V-cleavable circular aptamers of the present invention. Only one of six investigated exemplary DNA polymerases, particularly Bst DNA polymerase, was not inactivated by circular aptamer SEQ ID NO:12. Other DNA polymerases showed an inactivation in the presence of this aptamer as well as gradual endonuclease V-induced activation, although the efficiency of both processes was variable among the individual DNA polymerases.

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The invention claimed is:
 1. A kit for activating an aptamer-inactivated DNA polymerase, comprising: an endonuclease V enzymatic activity; and a DNA polymerase-binding circular oligonucleotide aptamer cleavable by an endonuclease V enzymatic activity.
 2. The kit of claim 1, wherein the circular oligonucleotide aptamer comprises one or more deoxyinosine nucleotides.
 3. The kit of claim 1, wherein the circular oligonucleotide aptamer comprises a stem-loop structure.
 4. The kit of claim 3, wherein the one or more deoxyinosine nucleotides are located in the stem segment of the stem-loop structure.
 5. The kit of claim 3, wherein the one or more deoxyinosine nucleotides are located in the loop segment of the stem-loop structure.
 6. The kit of claim 5, wherein the loop of the stem-loop structure, comprises a nucleotide sequence selected from the group consisting of 5′-TTCITAGCGTTT-3′ (SEQ ID NO:19), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20), 5′-TTCTTAICGTTT-3′ (SEQ ID NO:21), 5′-TTCIIAGCGTTT-3′ (SEQ ID NO:22), 5′-TTCITAICGTTT-3′ (SEQ ID NO:23), 5′-TTCTIAICGTTT-3′ (SEQ ID NO:24), and 5′-TTCIITAICGTTT-3′ (SEQ ID NO:25).
 7. The kit of claim 6, wherein the loop of the stem-loop structure comprises one of the nucleotide sequences 5′-TTCITAGCGTTT-3′ (SEQ ID NO:19), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20), or 5′-TTCTTAICGTTT-3′ (SEQ ID NO:21).
 8. The kit of claim 1, wherein the endonuclease V enzymatic activity comprises Thermotoga maritima endonuclease V enzymatic activity.
 9. The kit of claim 1, further comprising a DNA polymerase to provide a reaction mixture, wherein the endonuclease V-cleavable circular oligonucleotide aptamer binds reversibly to the DNA polymerase, wherein the circular oligonucleotide aptamer is present in an amount effective to inhibit DNA synthesis activity of the DNA polymerase in the reaction mixture, and wherein the endonuclease V enzymatic activity is capable of cleaving the circular oligonucleotide aptamer to reduce or eliminate binding of the circular oligonucleotide aptamer to the DNA polymerase, thereby activating or increasing the DNA synthesis activity of the DNA polymerase.
 10. The kit of claim 9, wherein the DNA polymerase activity and/or the endonuclease V enzymatic activity is temperature-dependent.
 11. The kit of claim 10, wherein the endonuclease V enzymatic activity increases when the reaction mixture is heated from a first temperature value to a second temperature value that promotes the endonuclease V enzymatic activity.
 12. The kit of claim 9, wherein the DNA polymerase, circular oligonucleotide aptamer, and endonuclease V enzymatic activity are in a dried state.
 13. The kit of claim 9, wherein the circular oligonucleotide aptamer comprises one or more deoxyinosine nucleotides.
 14. The kit of claim 13, wherein the circular oligonucleotide aptamer comprises a stem-loop structure.
 15. The kit of claim 14, wherein the one or more deoxyinosine nucleotides are located in the stem segment of the stem-loop structure.
 16. The kit of claim 14, wherein the one or more deoxyinosine nucleotides are located in the loop segment of the stem-loop structure.
 17. The kit of claim 16, wherein the loop of the stem-loop structure, comprises a nucleotide sequence selected from the group consisting of 5′-TTCITAGCGTTT-3′ (SEQ ID NO:19), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20), 5′-TTCTTAICGTTT-3′ (SEQ ID NO:21), 5′-TTCIIAGCGTTT-3′ (SEQ ID NO:22), 5′-TTCITAICGTTT-3′ (SEQ ID NO:23), 5′-TTCTIAICGTTT-3′ (SEQ ID NO:24), and 5′-TTCIITAICGTTT-3′ (SEQ ID NO:25.
 18. The kit of claim 17, wherein the loop of the stem-loop structure comprises one of the nucleotide sequences 5′-TTCITAGCGTTT-3′ (SEQ ID NO:19), 5′-TTCTIAGCGTTT-3′ (SEQ ID NO:20), or 5′-TTCTTAICGTTT-3′ (SEQ ID NO:21).
 19. The kit of claim 9, wherein the endonuclease V enzymatic activity comprises Thermotoga maritima endonuclease V enzymatic activity.
 20. The kit of claim 9, wherein the mixture further comprises one or more of dATP, dCTP, dGTP, and/or dTTP, and/or Mg²⁺ ion. 